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Abstract:

Antifungal compounds, an antifungal compound extracted from Epicoccum
purpurascens, also known as Epicoccum nigrum, methods of producing the
antifungal compounds, isolates and compositions comprising the antifungal
compounds, and methods of using the antifungal compounds.

Claims:

1-37. (canceled)

38. A biologically pure culture of Epicoccum purpurascens strain SVB-F1
on deposit at the National Measurement Institute, Australia (NMI) under
accession number V10/000331, deposited 18 Mar. 2010, or a culture having
the identifying characteristics thereof, or a culture extract or isolate
obtained therefrom.

40. The compound of claim 39 wherein R1 is selected from or is
substituted with alkylcarbonylalkyl, alkylcarbonyloxyalkyl,
alkyloxyalkyl, alkylsulfonyl, alkylsulfinyl, alkylthioalkyl,
hydroxyalkyl, mono-or di(alkyl)aminoalkyl, or wherein R1 is
hydrogen, hydroxyl, or a C1 to C30 alkyl or alkoxy group
optionally comprising one or more double bonds, and optionally
substituted with one or more groups selected from lower alkyl, lower
alkenyl, lower alkoxy, hydroxyl and carboxyl, or wherein R1 is a
C1 to C30 alkyl or alkoxy group comprising one or more double
bonds, and substituted with one or more groups selected from lower alkyl,
lower alkenyl, lower alkoxy, hydroxyl and carboxyl.

45. The compound of claim 42 wherein each R6 is independently
selected from hydrogen, methyl, methoxy, hydroxyl and carboxyl.

46. The compound of claim 43 wherein each R6 is independently
selected from hydrogen, methyl, methoxy, hydroxyl and carboxyl.

47. The compound of claim 39 wherein the compound is of Formula (IIIA)
##STR00015## wherein R2, R3, R4, and R5
areindependently selected from hydrogen, methyl, methoxy and hydroxyl;
and each R6 is independently selected from hydrogen, methyl,
methoxy, hydroxyl and carboxyl.

48. The compound of claim 39 wherein the compound is of Formula (IV)
##STR00016##

49. An isolate obtained or obtainable from Epicoccum purpurascens
comprising at least about 1 to 99% by weight of a compound of Formula (I)
or a salt thereof.

50. A composition comprising a compound of Formula (I) or a salt,
solvate, or hydrate thereof or an isolate obtained or obtainable from
Epicoccum purpurascens comprising at least about 1 to 99% by weight of a
compound of Formula (I) or a salt thereof and an agriculturally or
pharmaceutically acceptable carrier.

51. A composition comprising Epicoccum purpurascens strain SVB-F1 on
deposit at the National Measurement Institute, Australia (NMI) under
accession number V10/000331 or a culture having the identifying
characteristics thereof or an extract or isolate thereof, and an
agriculturally or pharmaceutically acceptable carrier.

52. A method of treating or preventing a fungal infection comprising
administration of: (a) a compound of Formula (I) or a salt, solvate, or
hydrate thereof; or (b) an isolate obtained or obtainable from Epicoccum
purpurascens comprising at least about 1 to 99% by weight of a compound
of Formula (I) or a salt thereof; (c) a composition comprising a compound
of Formula (I) or a salt, solvate, or hydrate thereof or an isolate
obtained or obtainable from Epicoccum purpurascens comprising at least
about 1 to 99% by weight of a compound of Formula (I) or a salt thereof
and a pharmaceutically acceptable carrier; or (d) a composition
comprising Epicoccum purpurascens strain SVB-F1 on deposit at the
National Measurement Institute, Australia (NMI) under accession number
V10/000331 or a culture having the identifying characteristics thereof or
an extract or isolate thereof, and a pharmaceutically acceptable carrier;
(e) or any combination of two or more of (a) to (d) above; to a subject
in need thereof.

53. A method of treating or preventing a fungal infection comprising
application of a. a compound of Formula (I) or a salt, solvate, or
hydrate thereof; or b. an isolate obtained or obtainable from Epicoccum
purpurascens comprising at least about 1 to 99% by weight of a compound
of Formula (I) or a salt thereof; c. a composition comprising a compound
of Formula (I) or a salt, solvate, or hydrate thereof or an isolate
obtained or obtainable from Epicoccum purpurascens comprising at least
about 1 to 99% by weight of a compound of Formula (I) or a salt thereof
and an agriculturally or pharmaceutically acceptable carrier; or d. a
composition comprising Epicoccum purpurascens strain SVB-F1 on deposit at
the National Measurement Institute, Australia (NMI) under accession
number V10/000331 or a culture having the identifying characteristics
thereof or an extract or isolate thereof, and an agriculturally or
pharmaceutically acceptable carrier; e. or any combination of two or more
of (a) to (d) above; to a target surface in need thereof.

54. The method of claim 52 wherein the fungal infection comprises
infection by a filamentous fungus.

56. The method of claim 52 wherein the composition comprises a
reproductively viable form and amount of Epicoccum purpurascens strain
SVB-F1 on deposit at the National Measurement Institute, Australia (NMI)
under accession number V10/000331 or a culture having the identifying
characteristics thereof in addition to a compound of Formula (I) or a
salt, solvate, or hydrate thereof.

57. A method of producing a compound of Formula (IV) or a salt thereof
##STR00017## the method comprising (a) culturing an organism of the
genus Epicoccum, preferably Epicoccum purpurascens in a culture medium to
produce the compound of Formula (IV) or salt thereof, and (b) optionally
extracting the compound of Formula (IV) or salt thereof from the culture
medium.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to antifungal compounds, an
antifungal compound extracted from Epicoccum purpurascens, also known as
Epicoccum nigrum. The invention also relates to methods of producing the
antifungal compounds, isolates and compositions comprising the antifungal
compounds, and methods of using the antifungal compounds.

BACKGROUND

[0002] Synthetic fungicides have been used as the main tool to control
fungal infection on growing and harvested crops. It has been estimated
that over 23 million kilograms of these synthetic fungicides are used
annually worldwide and it is felt that production and marketing of fruit
and vegetables would not be possible without their use (Tripathi and
Dubey, 2004).

[0003] The use of such chemicals has increased consumer concern and their
use is becoming more and more restricted due to toxicity problems and
environmental poIlution. This there is an increasing interest in finding
useful alternatives to chemical fungicides that are safe and have
negligible risk to human health and the environment.

[0004] Among these strategies, natural products with antifungal activity
are attractive because they are readily biodegradable and, therefore,
could be less toxic to the environment and consumers.

[0005] Therefore it is an object of the present invention to provide an
improved or alternative antifungal compound or to at least provide the
public with a useful choice.

SUMMARY OF THE INVENTION

[0006] Accordingly, in a first aspect the invention relates to an isolated
or substantially pure compound of Formula (I) or a salt, solvate, or
hydrate thereof

##STR00001##

wherein

[0007] R1 is hydrogen, hydroxyl, or a C1 to C30 alkyl or
alkoxy group optionally comprising one or more double bonds, optionally
comprising one or more triple bonds, and optionally substituted with one
or more groups selected from hydroxyl, halogen, cyano, nitro, amino,
alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl,
--NRaRb, --NRaC(═O)Rb,
--NRaC(═O)NRaRb, --NRaC(═O)ORb,
--NRaSO2Rb, ORa, --C(═O)Ra,
--C(═O)ORa, --C(═O)NRaRb,
--OC(═O)NRaRb, --SHa, --SRa, --SORa,
--S(═O)2Ra, --OS(═O)2Ra,
--S(═O)20Ra, wherein Ra and Rb are the same or
different and independently selected from the group comprising hydrogen,
halogen, alkyl, haloalkyl, thioalkyl, and substituted alkyl; and

[0009] In a second aspect the invention relates to an isolate obtained or
obtainable from Epicoccum purpurascens comprising at least about 1 to 99%
by weight of a compound of Formula (I) or a salt thereof.

[0010] In a third aspect the invention relates to a composition comprising
a compound of Formula (I) or a salt, solvate, or hydrate thereof or an
isolate obtained or obtainable from Epicoccum purpurascens comprising at
least about I to 99% by weight of a compound of Formula (I) or a salt
thereof and an agriculturally or pharmaceutically acceptable carrier.

[0011] In a fourth aspect the invention relates to a method of treating or
preventing a fungal infection comprising administration of a compound or
isolate of the invention to a subject in need thereof.

[0012] In a fifth aspect the invention relates to a method of treating or
preventing a fungal infection comprising application of a compound or
isolate of the invention to a target surface in need thereof, such as a
plant or its surroundings.

[0013] In a sixth aspect the invention relates to a method of producing a
compound of Formula (IV) or a salt thereof

##STR00002##

[0014] the method comprising

[0015] (a) culturing an organism of the
genus Epicoccum, preferably Epicoccum purpurascens in a culture medium to
produce the compound of Formula (IV) or salt thereof, and

[0016] (b)
optionally extracting the compound of Formula (IV) or salt thereof from
the culture medium.

[0017] In a seventh aspect the invention relates to a biologically pure
culture of Epicoccum purpurascens strain SVB-F1 on deposit at the
National Measurement Institute, Australia (NMI) under accession number
V10/000331, deposited 18 Mar. 2010, or a culture having the identifying
characteristics thereof.

[0018] In an eighth aspect the invention relates to a method of treating
or preventing a fungal infection comprising administering Epicoccum
purpurascens strain SVB-F1 or a culture having identifying
characteristics thereof or a composition comprising Epicoccum
purpurascens strain SVB-F1 or a culture having the identifying
characteristics thereof to a subject in need thereof.

[0019] In a ninth aspect, the invention relates to a method of treating or
preventing a fungal infection comprising applying Epicoccum purpurascens
strain SVB-F1 or a culture having the identifying characteristics thereof
or a composition comprising Epicoccum purpurascens strain SVB-F1 or a
culture having the identifying characteristics thereof to a target
surface in need thereof.

[0020] The following embodiments may relate to any of the above aspects.

[0021] In one embodiment of a compound of Formula (I), R1 is selected
from or is substituted with alkylcarbonylalkyl, alkylcarbonyloxyalkyl,
alkyloxyalkyl, alkylsulfonyl, alkylsulfinyl, alkylthioalkyl,
hydroxyalkyl, mono-or di(alkyl)aminoalkyl.

[0022] In one embodiment of a compound of Formula (I), R1 is
hydrogen, hydroxyl, or a C1 to C30 alkyl or alkoxy group
optionally comprising one or more double bonds, and optionally
substituted with one-or more groups selected from lower alkyl, lower
alkenyl, lower alkoxy, hydroxyl and carboxyl.

[0023] In one embodiment of a compound of Formula (I), R1 is a
C1 to C30 alkyl or alkoxy group comprising one or more dOuble
bonds, and substituted with one or more groups selected from lower alkyl,
lower alkenyl, lower alkoxy, hydroxyl and carboxyl.

[0024] In one embodiment of a compound of Formula (I), R2, R3,
R4, and R5 are independently selected from hydrogen, alkyl,
substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, and halogen, or
are independently selected from hydrogen, methyl, methoxy and hydroxyl.
In another embodiment of a compound of Formula (I), R2, R3,
R4, and R5 are independently selected from hydrogen and
hydroxyl.

[0025] In one embodiment the compound of Formula I comprises a compound of
Formula (II)

##STR00003##

wherein R2, R3, R4, and R5 are as defined above;

[0026] each is independently a single bond, a double bond or a triple
bond; and

[0028] In one embodiment of a compound of Formula (II), each is
independently a single bond or a double bond (cis or trans), or each is
a double bond. In one embodiment of a compound of Formula (II), R2,
R3, R4, and R5 are independently selected from hydrogen,
alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, and
halogen, or are independently selected from hydrogen, methyl, methoxy and
hydroxyl. In another embodiment of a compound of Formula (II), R2,
R3, R4, and R5 are independently selected from hydrogen
and hydroxyl. In one embodiment of a compound of Formula (II), each
R6 is independently selected from hydrogen, methyl, methoxy,
hydroxyl and carboxyl. In another embodiment of a compound of Formula
(II), each R6 is independently selected from hydrogen, methyl, and
carboxyl. In another embodiment the compound of Formula I comprises a
compound of Formula

##STR00004##

wherein

[0029] R2, R3, R4, and R5 are as defined above;

[0030] each is independently a single bond, a double bond or a triple
bond; and

[0032] In one embodiment of a compound of Formula (III), each is
independently a single bond or a double bond (cis or trans), or each is
a double bond. In one embodiment of a compound of Formula (III), R2,
R3, R4, and R5 are independently selected from hydrogen,
alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, and
halogen, or are independently selected from hydrogen, methyl, methoxy and
hydroxyl. In another embodiment of a compound of Formula (III), R2,
R3, R4, and R5 are independently selected from hydrogen
and hydroxyl. In one embodiment of a compound of Formula (III), each
R6 is independently selected from hydrogen, methyl, methoxy,
hydroxyl and carboxyl. In another embodiment of a compound of Formula
(III), each R6 is independently selected from hydrogen, methyl, and
carboxyl.

[0033] In one embodiment the compound of Formula I comprises a compound of
Formula (IIIA)

##STR00005##

wherein

[0034] R2, R3, R4, and le are independently selected from
hydrogen, methyl, methoxy and hydroxyl; and

[0035] each R6 is independently selected from hydrogen, methyl,
methoxy, hydroxyl and carboxyl.

[0036] In yet another embodiment the compound of Formula I comprises a
compound of Formula (IV)

##STR00006##

[0037] In one embodiment the compound of Formula (I) to (IV) is obtained
or obtainable from an organism of the genus Epicoccum. In another
embodiment the compound of Formula (I) to (IV) is obtained or obtainable
from Epicoccum purpurascens. In another embodiment the compound of
Formula (I) to (IV) is obtained or obtainable from Epicoccum purpurascens
strains SVB-F1 (V10/000331), ICMP2048, ICMP2931, ICMP10458, ICMP10459,
ICMP10460, ICMP11503, ICMP15700, ICMP15816 or ICMP16305.

[0038] In one embodiment the fungus comprises a filamentous fungus and the
fungal infection comprises a filamentous fungal infection.

[0039] In another embodiment the fungus comprises a filamentous fungus
that is an animal pathogen and the fungal infection comprises a
filamentous fungal infection of animals.

[0040] In yet another embodiment the fungus comprises a phytopathogenic
filamentous fungus and the fungal infection comprises a phytopathogenic
filamentous fungal infection.

[0047] In one embodiment the isolate obtained or obtainable from Epicoccum
purpurascens comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% by
weight of a compound of Formula (I) or a salt thereof, and useful ranges
may be selected between any of these values (for example, about 1 to
about 99, about 5 to about 99, about 10 to about 99, about 15 to about
99, about 20 to about 99, about 25 to about 99, about 30 to about 99,
about 35 to about 99, about 40 to about 99, about 45 to about 99, about
50 to about 99, about 55 to about 99, about 60 to about 99, about 65 to
about 99, about 70 to about 99, about 75 to about 99, about 80 to about
99, about 85 to about 99, or about 90 to about 99% by weight).

[0048] It should be understood that any isolates useful herein include
isolates obtained or obtainable from a culture comprising Epicoccum
purpurascens or a culture from which Epicoccum purpurascens has been
removed.

[0049] In one embodiment the method of treating or preventing the fungal
infection comprises preventing or decreasing fungal growth or preventing
or decreasing spore germination or both.

[0050] In one embodiment a composition useful herein comprises at least
about , 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95 or 100 mg/mL of a compound of Formula (I) to (IV),
preferably a compound of Formula (IV), and useful ranges may be selected
between any of these values (for example, about 0.01 to about 1.0, about
0.01 to about 10, about 0.01 to about 20, about 0.01 to about 30, about
0.01 to about 40, about 0.01 to about 50, about 0.01 to about 60, about
0.01 to about 70, about 0.01 to about 80, about 0.01 to about 90, about
0.01 to about 100, about 0.1 to about 1.0, about 0.1 to about 10, about
0.1 to about 20, about 0.1 to about 30, about 0.1 to about 40, about 0.1
to about 50, about 0.1 to about 60, about 0.1 to about 70, about 0.1 to
about 80, about 0.1 to about 90, about 0.1 to about 100, about 0.7 to
about 1.0, about 0.7 to about 10, about 0.7 to about 20, about 0.7 to
about 30, about 0.7 to about 40, about 0.7 to about 50, about 0.7 to
about 60, about 0.7 to about 70, about 0.7 to about 80, about 0.7 to
about 90, or about 0.7 to about 100 mg/mL).

[0051] In various embodiments the composition useful herein comprises a
reproductively viable form and amount of Epicoccum purpurascens SVB-F1.

[0052] In one embodiment, the composition comprises a reproductively
viable form and amount of Epicoccum purpurascens SVB-F1 and one or more
compounds or isolates of the invention.

[0053] In one embodiment a composition useful herein may comprise one or
more additional agricultural agents. In another embodiment a method of
the invention may comprise separate, simultaneous or sequential
administration of the compound of Formula (I) to (IV) or the isolate and
one or more additional agricultural agents. In one embodiment the
additional agricultural agent is selected from one or more fungicides,
one or more fertilizers, one or more antibiotics, one or more antivirals
or one or more insecticides. In one embodiment the compound of Formula
(I) to (IV) or the isolate can be placed on the target surface before,
after or simultaneously with the other agricultural agent.

[0055] In one embodiment the culture medium comprises a source of nitrate,
a source of protein, a source of histidine, or a source of protein
comprising histidine, or any combination of any two or more thereof.

[0056] In this specification where reference has been made to patent
specifications, other external documents, or other sources of
information, this is generally for the purpose of providing a context for
discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as
an admission that such documents, or such sources of information, in any
jurisdiction, are prior art, or form part of the common general knowledge
in the art.

[0057] It is intended that reference to a range of numbers disclosed
herein (for example, 1 to 10) also incorporates reference to all rational
numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,
7, 8, 9 and 10) and also any range of rational numbers within that range
(for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all
sub-ranges of all ranges expressly disclosed herein are hereby expressly
disclosed. These are only examples of what is specifically intended and
all possible combinations of numerical values between the lowest value
and the highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.

[0058] To those skilled in the art to which the invention relates, many
changes in construction and differing embodiments and applications of the
invention will suggest themselves without departing from the scope of the
invention as defined in the appended claims. The disclosures and the
descriptions herein are purely illustrative and are not intended to be in
any sense limiting.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The inventors have surprisingly discovered that Epicoccuin
purpurascens (also known as Epicoccuin nigrum) produces an antifungal
compound having the following formula and that the compound is able to
treat or prevent infections by filamentous fungi.

##STR00007##

1. Definitions

[0060] The term "agriculturally acceptable" is intended to include any
material, such as a carrier, that may used in agriculture and that
preferably aids application of a compound or composition of the invention
to the intended target surface or that aids storage, transport or
handling. Agriculturally acceptable carriers used in compositions for
application to plants and plant material are preferably non-phytotoxic or
only mildly phytotoxic. A suitable carrier may be a solid, liquid or gas
depending on the desired formulation. In one embodiment preferred
carriers include polar liquid carriers including but.not limited to
water, alcohol, mineral oil and vegetable oil. Agriculturally acceptable
salts include those that substantially maintain the desired activity of a
compound of the invention while also being acceptable for use in
agriculture and preferably being non-phytotoxic or only mildly
phytotoxic.

[0061] The term "alkenyl" means a hydrocarbon radical having at least one
double bond including, but not limited to, ethenyl, propenyl, 1-butenyl,
2-butenyl and the like.

[0062] The term "alkoxy" means an O-alkyl group wherein "alkyl" is as
defined herein, for example, methoxy, ethoxy, and the like.

[0063] The term "alkyl" means a straight or branched chain, noncyclic or
cyclic hydrocarbon radical, which may be fully saturated, mono- or
polyunsaturated, including di- and multivalent radicals, and may have the
number of carbon atoms designated (for example, C1-C10 means
one to ten carbon atoms). Examples of saturated straight chain alkyls
include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl and the like.
Examples of saturated branched chain alkyls include isopropyl, isobutyl,
sec-butyl, test-butyl, isopentyl and the like. Representative saturated
cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
and the like. An unsaturated alkyl group is one having one or more double
or triple bonds. Examples of unsaturated straight chain alkyl groups
include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),
2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,
3-butynyl, and the higher homologs and isomers. The term "alkyl," unless
otherwise stated, is also meant to include those derivatives of alkyl
defined in more detail below as "alkenyl", "alkynyl", "cycloalkyl" and
"alkylene."

[0064] Theterms "alkylamino" and "dialkyl amino" means one alkyl group or
two alkyl groups, respectively, attached through a nitrogen bridge (i.e.
--N-alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino,
and the like.

[0065] The term "alkylcarbonylalkyl," means an alkyl substituted with a
--C(═O)alkyl group.

[0066] The term "alkylcarbonyloxyalkyl," means an alkyl substituted with a
--C(═O)Oalkyl group or a --OC(═O)alkyl group.

[0067] The term "alkyloxyalkyl," means an alkyl substituted with an
--O-alkyl group.

[0068] The term "alkylene" means a divalent radical derived from an
alkane, as exemplified by --CH2CH2CH2CH2--.
Typically, an alkylene group will have from 1 to 24 carbon atoms.

[0069] The term "alkylsulfonyl," means an alkyl group attached through a
sulfonyl bridge (i.e. --SO2-alkyl) such as methylsulfonyl,
ethylsulfonyl, and the like.

[0070] The term "alkylsulfinyl" means an alkyl group attached through a
sulfinyl bridge (i.e. S(O)-alkyl) such as methylsulfinyl, ethylsulfinyl,
propylsulfinyl, butylsulfinyl, and the like.

[0071] The term "alkylthioalkyl" means an alkyl substituted with a
--S-alkyl group.

[0072] The term "alkynyl" means a hydrocarbon radical having at least one
triple bond including, but not limited to, ethynyl, propynyl, 1-butynyk
2-butynyl and the like.

[0073] The term "antifungal" means an ability to antagonise one or more
fungi by preventing or controlling a fungal infection as described below.
Accordingly an antifungal agent, such as an antifungal compound, isolate
or composition, is an agent that is an antagonist of one or more fungi.
Such an agent is herein considered to have antifungal activity.

[0074] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting each statement in
this specification that includes the term "comprising", features other
than that or those prefaced by the term may also be present. Related
terms such as "comprise" and "comprises" are to be interpreted in the
same manner.

[0075] The term "cycloalkyl" means a cyclic version of "alkyl", and
includes di-and poly-homocyclic rings such as decalin and adamentane.
Examples of cycloalkyls include cyclopentyl, cyclohexyl, 1-cyclohexenyl,
3-cyclohexenyl, cycloheptyl, and the like.

[0076] The terms "halo" or "halogen" means a fluorine, chlorine, bromine,
or iodine atom. Additionally, terms such as "fluoroalkyl," are meant to
include monofluoroalkyl and polyfluoroalkyl.

[0077] The term "haloalkyl" means an alkyl group having at least one
hydrogen atom replaced with a halogen, for example trifluoromethyl, and
the like.

[0078] The term "lower" means a group having 1 to 8 carbon atoms, for
example, "lower alkyl", "lower alkenyl", "lower alkynyl", "lower
alkylene" and "lower alkoxy" mean an alkyl, alkenyl, alkynyl, alkylene or
alkoxy group having 1 to 8 carbon atoms.

[0079] The term "hydroxyalkyl" means an alkyl substituted with at least
one hydroxyl group.

[0080] The term "mono-or di(alkyl)aminoalkyl" means an alkyl substituted
with a mono- or di(alkyl)amino.

[0081] The term "pharmaceutically acceptable" as used herein refers to
carriers, diluents, excipients, compounds, ingredients, materials,
compositions, dosage forms and the like, that are within the scope of
sound medical judgment, suitable for use in contact with the tissues of
the subject in question (e.g. human) without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio. Each carrier, diluent,
excipient, and the like, must also be "acceptable" in the sense of being
compatible with the other ingredients of the formulation.

[0082] The term "plant" means whole plants and includes to plant parts,
cuttings as well as plant products including roots, leaves, flowers,
seeds, sterns, callus tissue, nuts and fruit, bulbs, tubers, corms,
grains, cuttings, root stock, or scions, and includes any plant material
whether pre-planting, during growth, and at or post harvest. Plants that
may benefit from the application of the present invention cover a broad
range of agricultural and horticultural crops. The compounds, isolates
and compositions of the invention are also suitable for application in
organic production systems.

[0083] A "strain having the identifying characteristics of [a specified
strain]", including a homologue or mutant of the specified strain, is
closely related to (i.e., shares a common ancestor with) or derived from
the specified strain, but will usually differ from the specified strain
in one or more genotypic or phenotypic characteristics. Mutants are
generally identifiable through assessment of genetic differences.
Homologues are identifiable through assessment of the degree of genetic,
biochemical and morphological difference and use of taxonomic methods,
including for example analyses such as cladistics. However, a strain
having the identifying characteristics of [a specified strain], including
a homologue or mutant of the specified strain will retain antifungal
efficacy, will be distinguishable from other bacterial strains, and will
be identifiable as a homologue or mutant of the parent strain using the
techniques described above.

[0084] The term "substituted" as used herein with reference to any of the
herein defined groups (e.g. alkyl, etc.) means a group or compound
wherein at least one hydrogen atom has been replaced by a chemical
substituent. In the case of a keto substituent (--(C═O)--) two
hydrogen atoms are replaced.

[0086] The term "subject" is intended to include any animal or target
surface that is in need of the treatment or prevention of fungal
infection or fungal contamination. Animal subjects include mammals,
particularly humans, livestock animals such as cows, sheep, pigs and
goats, and companion animals such as horses, dogs and cats.

[0087] The term "surroundings" when used in reference to a plant subject
includes soil, water, leaf litter, or growth media adjacent to or around
the plant or the roots, tubers or the like thereof, adjacent plants,
cuttings of said plant, supports, water to be administered to the plant,
and coatings including seed coatings. It further includes storage,
packaging or processing materials such as protective coatings, boxes and
wrappers, and planting, maintenance or harvesting equipment.

[0088] The term "target surface" to which a compound or composition of the
invention may be applied includes but is not limited to plants and plant
surroundings, plant material including but not limited to roots, bulbs,
tubers, corms, leaves, flowers, seeds, stems, callus tissue, nuts,
grains, fruit, cuttings, root stock, scions, harvested crops including
but not limited to roots, bulbs, tubers, corms, leaves, flowers, seeds,
stems, callus tissue, nuts, grains, fruit, cuttings, root stock, scions,
or any surface that may contact harvested crops including but not limited
to harvesting equipment, packaging equipment and packaging material.

[0089] The term "treating or preventing a fungal infection" is intended to
include preventing or controlling a fungal infection and "controlling" is
intended to mean at least maintaining, preferably maintaining or
reducing, and more preferably reducing the degree of infection by a
fungal pathogen including but not limited to the pathogens listed herein.
In one embodiment, "controlling a fungal infection" means the compound,
isolate or composition is able to substantially eradicate an existing
fungal infection.

[0090] The term "thioalkyl" means an alkyl group attached through a sulfur
bridge (i.e. --S-alkyl) such as methylthio, ethylthio, and the like.

2. Compounds Obtainable from Epicoccum purpurascens

[0091] The inventors believe compounds that are structurally related to
the compound of Formula (IV) are likely to have similar antifungal
activity, including antifungal activity against filamentous fungi,
including fungi that are phytopathogens or animal pathogens. Accordingly,
the invention relates to a compound of Formula (I) or a salt, solvate, or
hydrate thereof

##STR00008##

wherein

[0092] R1 is hydrogen or a C1 to C30 alkyl group optionally
comprising one or more double bonds, optionally comprising one or more
triple bonds, and optionally substituted with one or more groups selected
from hydroxyl, halogen, cyano, nitro, amino, alkylamino, dialkylamino,
alkyl, alkoxy, alkylthio, haloalkyl, --NRaRb,
--NRaC(═O)Rb, --NRaC(═O)NRaRb,
--NRaC(═O)ORb, --NRaSO2Rb, ORa,
--C(═O)Ra, --C(═O)ORa, --C(═O)NRaRb,
--OC(═O)NRaRb, --SH, --SRa, --SORa,
--S(═O)2Ra, --OS(═O)2Ra,
--S(═O)2ORa, wherein Ra and Rb are the same or
different and independently selected from the group comprising hydrogen,
halogen, hydroxyl, halogen, alkyl, haloalkyl, thioalkyl, and substituted
alkyl; and

[0094] In various embodiments the invention also relates to compounds of
Formula (II), (III) and (IIIA) described above.

[0095] In one embodiment the compound of Formula (I) is the compound of
Formula (IV)

##STR00009##

[0096] It should be understood that a certain compound may exist in one or
more particular geometric, optical, enantiomeric, diasteriomeric,
epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms,
including but not limited to, cis- and trans-forms; E- and Z-forms; c-,
t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and
L-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; α- and
β-forms; axial and equatorial forms; boat-, chair-, twist-,
envelope-, and halfchair-forms; and combinations thereof, hereinafter
collectively referred to as "isomers" (or "isomeric forms").

[0097] Some compounds of Formula (I) to (IV) have at least one
asymmetrical carbon atom and therefore all isomers, including
enantiomers, stereoisomers, rotamers, tautomers and racemates of the
compounds are contemplated as being part of this invention. The invention
includes D and L isomers in both pure form and in admixture, including
racemic mixtures. Isomers can be prepared using conventional techniques,
either by reacting optically pure or optically enriched starting
materials or by separating isomers of a compound of the invention.
Isomers may also include geometric isomers, e.g., when a double bond is
present.

[0098] Note that, except as discussed below for tautomeric fomis,
specifically excluded from the term "isomers," as used herein, are
structural (or constitutional) isomers (i.e., isomers that differ in the
connections between atoms rather than merely by the position of atoms in
space). For example, a reference to a methoxy group, --OCH3, is not
to be construed as a reference to its structural isomer, a hydroxymethyl
group, --CH2OH. However, a preference to a class of structures may
well include structurally isomeric forms falling within that class (e.g.,
C1-7 alkyl includes /?-propyl and iso-propyl; butyl includes iso-,
sec-, and tet-butyl).

[0099] The above exclusion does not relate to tautomeric forms, for
example, keto-, enol-, and enolate-forms, as in, for example, the
following tautomeric pairs: keto/enol, imine/enamine, amide/imino
alcohol, amidine/amidine nitroso/oxime, thioketone/enethiol, N-
nitroso/hyroxyazo, and nitro/aci-nitro.

[0100] Note that specifically included in the term "isomer" are compounds
with one or more isotopic substitutions. For example, H may be in any
isotopic form, including 1H, 2H (D), and 3H (T); C may be
in any isotopic form, including 12C, 13C, and 14C; O may
be in any isotopic form, including 16O and 18O; and the like.

[0101] Unless otherwise specified, a reference to a particular compound
includes all such isomeric forms, including racemic and other mixtures
thereof. Methods for the preparation (e.g., asymmetric synthesis) and
separation (e.g., fractional crystallisation and chromatographic means)
of such isomeric forms are either known in the art or are readily
obtained by adapting the methods taught herein in a known manner.

[0102] Also unless otherwise specified, a reference to a particular
compound also includes ionic, salt, solvate, hydrate, and protected forms
of the compound.

[0103] If the compound is cationic, or has a functional group which may be
cationic (e.g., --NH-- may be --NH3.sup.+), then a salt may be
formed with a suitable anion. Examples of suitable inorganic anions
include, but are not limited to, those derived from the following
inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric,
sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of
suitable organic anions include, but are not limited to, anions from the
following organic acids: acetic, propionic, succinic, gycolic, stearic,
lactic, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetyoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, and valeric.

[0104] It may be convenient or desirable to prepare, purify, or handle a
corresponding solvate of the active compound. The term "solvate" is used
herein in the conventional sense to refer to a complex of solute (e.g.,
active compound, salt of active compound) and solvent. If the solvent is
water, the solvate may be conveniently referred to as a hydrate, for
example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

[0105] It may be convenient or desirable to prepare, purify, or handle the
active compound in a chemically protected form. The term "chemically
protected form," as used herein, relates to a compound in which one or
more reactive functional groups are protected from undesirable chemical
reactions, that is, are in the form of a protected or protecting group
(also known as masked or masking group). By protecting a reactive
functional group, reactions involving other unprotected reactive
functional groups can be performed, without affecting the protected
group; the protecting group may be removed, usually in a subsequent step,
without substantially affecting the remainder of the molecule. See, for
example, Protective Groups in Organic J Synthesis (T. Green and P. Wuts,
Wiley, 1991), incorporated herein by reference.

3. Isolation of Compounds of Formula (IV) from Epicoccum purpurascens

[0106] The compound of Formula (IV) can be isolated from Epicoccum
purpurascens (also known as Epicoccum nigrum) by culturing the organism
and then extracting the compounds to the desired degree of purity.

[0107] Accordingly, in one embodiment the compound of Formula (I) to (IV)
is obtained or obtainable from an organism of the genus Epicoccum. In
another embodiment the compound of Formula (I) to (IV) is obtained or
obtainable from Epicoccum purpurascens. In a further embodiment the
compound of Formula (I) to (IV) is obtained or obtainable from Epicoccum
purpurascens strains SVB-F1 (V10/000331), ICMP2048, ICMP2931, ICMP10458,
ICMP10459, ICMP10460, ICMP11503, ICMP15700, ICMP15816 or ICMP16305. As
shown in the examples below, each of these strains produces the compound
of Formula (IV). The Epicoccum purpurascens strain SVB-F1 is on deposit
under accession number V10/000331 at the National Measurement Institute,
Australia, deposited on 18 Mar. 2010. The ICMP strains are available from
the International Collection of Microorganisms from Plants (ICMP)
administered by Landcare Research, New Zealand (see
http://nzfungi.landcareresearch.co.nz/icmp/search cultures.asp--last
accessed 23 Nov. 2009). Epicoccum purpurascens strains are also available
from the American Type Culture Collection (ATCC), including but not
limited to the strains ATCC 10999, ATCC 32948, ATCC 34417, ATCC 34547,
ATCC 34929, ATCC 44336, ATCC 46473, ATCC 46878, ATCC 46880, ATCC 46881,
ATCC 58875, ATCC 62191, and ATCC 66091. These strains discussed above may
also be used in the methods of culturing organisms of the genus Epicoccum
described herein.

[0108] The compound of Formula (IV) can be isolated from Epicoccum
purpurascens by culturing the organism under submerged conditions in
industrial bioreactors known in the art and then extracting the compounds
to the desired degree of purity using centrifugation or membrane
filtration or both to remove suspended solids, biomass, fungal spores and
soluble proteins (such as enzymes). Alternatively, the compound of
Formula (IV) can be isolated from Epicoccum puipurascens by culturing the
organism on a solid substrate using solid fermentation techniques known
in the art and then extracted with a suitable environmentally-friendly
and non-toxic solvent (including but not limited to methanol, ethanol,
ethyl acetate, for example) and further extracted to the desired degree
of purity using centrifugation or membrane filtration or both to remove
suspended solids, biomass, fungal spores and soluble proteins (such as
enzymes). In both alternatives the product of centrifugation or the
filtrate can be further purified if necessary by column chromatography
using acid silica or any other appropriate polar solid matrix. Other
suitable known alternatives for isolating the compounds will be apparent
to those skilled in the art.

[0109] The inventors have found that compounds of Formula (IV).may
usefully be produced by culturing an organism of the genus Epicoccum in a
culture medium comprising nitrate, protein or histidine (data not shown).
Accordingly, in one embodiment the culture medium comprises a source of
nitrate, a source of protein, a source of histidine, or a source of
protein comprising histidine, or any combination of any two or more
thereof. Examples of useful sources of nitrate include but are not
limited to nitrate salts, any agriculturally or pharmaceutically
acceptable source of nitrate, sodium nitrate, magnesium nitrate,
potassium nitrate and calcium nitrate, or any combination of any two or
more thereof, and other useful nitrate sources will be apparent to those
skilled in the art. Examples of useful sources of protein include but are
not limited to peptone, yeast extract, and potato extract, or any
combination of any two or more thereof, and other useful protein sources
will be apparent to those skilled in the art. Examples of useful sources
of histidine include but are not limited to histidine as a free amino
acid, and dipeptides, tripeptides, oligopeptides or polypeptides
comprising histidine, or any combination of any two or more thereof, and
other useful histidine sources will be apparent to those skilled in the
art.

4. Target Agricultural Pathogens

[0110] The compounds; isolates, are compositions of the invention are
useful to treat or prevent infections caused by filamentous fungi. In one
embodiment the fungus comprises a filamentous fungus and the fungal
infection comprises a filamentOus fungal infection.

[0114] Species from the above families and genus are responsible for many
agricultural plant diseases. For example, Botrytis cinera is responsible
for blossom blights and fruit rots, and Rhizoctonia Solani is responsible
for `wire stem`. Other species from the listed families and genus are
responsible for diseases such as `pitting disease`, `ryegrass blast` and
`southern blight`.

[0116] It should be understood that filamentous fungi are readily
identifiable as such by those skilled in the art and that the compounds,
isolates, compositions and methods of the invention are equally useful in
treating or preventing fungal infections caused by those other
filamentous fungi. Equally, the compounds, isolates and compositions of
the invention may be assayed for activity against any fungal species
according to the methods described in the examples below.

5. Target Animal Pathogens

[0117] In other embodiments the fungus comprises a filamentous fungus that
is an animal pathogen and the fungal infection comprises a filamentous
fungal infection of animals.

[0120] It should be understood that the compounds, isolates and
compositions of the invention may be assayed for activity against any
fungal species that is pathogenic to animals, particularly humans,
according to the methods described in the examples below.

6. Agricultural Compositions of the Invention

[0121] Compositions useful herein included any agriculturally or
pharmaceutically acceptable composition that can carry a compound of
Formula (I) to (IV) or a salt, solvate or hydrate thereof, or an isolate
of the invention, as well as compositions suitable for maintaining the
viability of Epicoccum purpurascens SVB-F1 or a strain having the
identifying characteristics of Epicoccum purpurascens SVB-F1. The
compositions of the invention, in various embodiments, are sprayable and
may be sprayed onto a subject in need thereof, or formulated as a
concentrate that is sprayable on addition of agriculturally acceptable
carriers and/or spray adjuvants. The compositions of the present
invention may also be a solid, such as a powder, that is placed on to the
target surface. Preferred compositions of the invention are shelf stable.
The term "shelf stable" is intended to mean that a composition of the
invention does not separate out into separate phases, develop offensive
odours and/or develop microbial growth.

[0122] Compositions of the invention may be formulated as, for example,
concentrates, solutions, sprays, aerosols, immersion baths, dips,
emulsions, wettable powders, soluble powders, suspension concentrates,
dusts, granules, water dispersible granules, microcapsules, pastes, gels
and other formulation types by well-established procedures. These
procedures include mixing and/or milling of the active ingredients with
agriculturally acceptable carrier substances, such as fillers, solvents,
excipients, surfactants, suspending agents, speaders/stickers
(adhesives), antifoaming agents, dispersants, wetting agents, drift
reducing agents, auxiliaries and adjuvants. Depending on the format
chosen, compositions may be formulated for methods of application such as
injection, rubbing or brushing, as are known in the art. Indirect
applications of the composition to the plant surroundings or environment
such as soil, water, or as seed coatings are potentially possible.

[0123] The compositions of the present invention may further comprise
components such as liquid or solid carriers, binders, fillers and other
agriculturally or pharmaceutically suitable additives.

[0126] In one embodiment the carrier may also be liquid, for example,
water; sugar solutions; alcohols, particularly butanol or glycol, as well
as their ethers or esters, particularly methylglycol acetate; ketones,
particularly acetone, cyclohexanone, methylethyl ketone,
methylisobutylketone, or isophorone; petroleum fractions such as
paraffinic or aromatic hydrocarbons, particularly xylenes or alkyl
naphthalenes; mineral or vegetable oils; aliphatic chlorinated
hydrocarbons, particularly trichloroethane or methylene chloride;
aromatic chlorinated hydrocarbons, particularly chlorobenzenes;
water-soluble or strongly polar solvents such as dimethylformamide,
dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; or the like
or a mixture thereof.

[0127] In one embodiment surfactants include nonionic surfactants, anionic
surfactants, cationic surfactants and/or amphoteric surfactants and
promote the ability of aggregates to remain in solution during spraying.

[0129] Wetting agents reduce surface tension of water in the composition
and thus increase the surface area over which a given amount of the
composition may be applied. Examples of wetting agents include but are
not limited to salts of polyacrylic acids, salts of lignosulfonic acids,
salts of phenolsulfonic or naphthalenesulfonic acids, polycondensates of
ethylene oxide with fatty alcohols or fatty acids or fatty esters or
fatty amines, substituted phenols (particularly alkylphenols or
arylphenols), salts of sulfosuccinic acid esters, taurine derivatives
(particularly alkyltaurates), phosphoric esters of alcohols or of
polycondensates of ethylene oxide with phenols, esters of fatty acids
with polyols, or sulfate, sulfonate or phosphate functional derivatives
of the above compounds.

[0130] In one embodiment the preferred method of applying the compound or
composition of the invention is to spray a dilute or concentrated
solution by handgun or commercial airblast.

[0131] A carrier which provides for slow or delayed release of a compound
of the invention may also be included in a composition of the invention.

[0132] The compositions of the present invention may be used alone or in
combination with one or more other agricultural agents,' including
pesticides, insecticides, acaracides, additional fungicides,.
bactericides, herbicides, antibiotics, antimicrobials, nemacides,
rodenticides, entomopathogens, pheromones, attractants, plant growth
regulators, plant hormones, insect growth regulators, chemosterilants,
microbial pest control agents, repellents, viruses, phagostimulents,
plant nutrients, plant fertilisers and biological controls. When used in
combination with other agricultural agents the administration of the two
agents may be separate, simultaneous or sequential. Specific examples of
these agricultural agents are known to those skilled in the art, and many
are readily commercially available.

[0134] Examples of antibiotics include but are not limited to
oxytetracyline and streptomycin.

[0135] Examples of fungicides include but are not limited to the following
classes of fungicides: carboxamides, benzimidazoles, triazoles,
hydroxypyridines, dicarboxamides, phenylamides, thiadiazoles, carbamates,
cyano-oximes, cinnamic acid derivatives, morpholines, imidazoles,
beta-methoxy acrylates and pyridines/pyrimidines.

[0136] Further examples of fungicides include but are not limited to
natural fungicides, organic fungicides, sulphur-based fungicides,
copper/calcium fungicides and elicitors of plant host defences.

[0137] Examples of natural fungicides include but are not limited to whole
milk, whey, fatty acids or esterified fatty acids.

[0138] Examples of organic fungicides include but are not limited to any
fungicide which passes an organic certification standard such as
biocontrol agents, natural products, elicitors (some of may also be
classed as natural products), and sulphur and copper fungicides (limited
to restricted use). An example of a sulphur-based fungicide is
Kumulus® DF (BASF, Germany). An example of a copper fungicide is
Kocide® 2000 DF (Griffin Corporation, USA). Examples of elicitors
include but are not limited to chitosan, Bion®, BABA
(DL-3-amino-n-butanoic acid,(3-aminobutyric acid) and Milsana®
(Western Farm Service, Inc., USA).

[0139] In some embodiments non-organic fungicides may be employed.
Examples of non-organic fungicides include but are not limited to
Bravo® (for control of PM on cucurbits); Supershield® (Yates, NZ)
(for control of Botrytis and PM on roses); Topas® 200EW (for control
of PM on grapes and cucurbits); Flint® (for control of PM on apples
and cucurbits); Amistar® WG (for control of rust and PM on cereals);
and Captan®, Dithane®, Euparen®, Rovral®, Scala®,
Shirlan®, Switch® and Teldor® (for control of Botrytis on
grapes).

[0141] An example of a biological control is the BotryZen® biological
control agent comprising Ulocladiwn oudemansii.

[0142] Confirmation of the ability of compositions of the inventionto
treat fungal growth may be obtained by inoculating plant material with a
target organism and then applying a compound, isolate or composition of
the invention. Efficacy is confirmed by a reduction in the degree of
growth or the disappearance of the target organism compared to an
untreated control.

7. Therapeutic Compositions of the Invention

[0143] A composition useful herein may be formulated as a food, drink,
food additive, drink additive, dietary supplement, nutritional product,
medical food, enteral or parenteral feeding product, meal replacement,
cosmeceutical, nutraceutical, or pharmaceutical, or as a coating or other
component of a medical device. Appropriate .formulations may be prepared
by an art skilled worker with regard to that skill and the teaching of
this specification. Compositions useful herein include any composition
that is able to carry a compound of Formula (I) to (IV) or an isolate of
the invention.

[0144] In one embodiment the composition is in the form of a powder, a
tablet, a caplet, a pill, a hard or soft capsule or a lozenge. In another
embodiment the composition is in the form of a cachet, a dispensable
powder, granules, a suspension, an elixir, a liquid, a drink, or any
other form that can be added to food or drink, including for example
water or fruit juice. In a further embodiment the composition is an
enteral product, a solid enteral product or a liquid enteral product. In
yet another embodiment, the composition is in the form of a cream,
ointment, paste, drop solution including eye drops or ear drops, inhaler,
inhalable composition, dressing, pad, or spray.

[0145] The compositions useful herein may be formulated to allow for
administration to a subject by any chosen route, including but not
limited to oral or nasal (including by inhalation), vaginal, rectal or
parenteral (including topical, subcutaneous, intramuscular and
intravenous) administration. Those skilled in the art will appreciate
that the route of administration to a subject will typically take into
account the purpose for which the composition is being administered for
example, where a pharmaceutical composition of the invention is being
administered to treat a disease or disorder, the route of administration
will typically be chosen taking into account the nature of the disease or
disorder. Accordingly, exemplary compositions for the treatment of skin
infections or infections of mucosal membranes may be formulated for
topical administration.

[0146] In general, for oral administration a dietary (a food, food
additive or food supplement for example), nutraceutical or pharmaceutical
composition useful herein may be formulated by a skilled worker according
to known formulation techniques. Thus, a pharmaceutical composition
useful according to the invention may be formulated with an appropriate
pharmaceutically acceptable carrier (including excipients, diluents,
auxiliaries, and combinations thereof) selected with regard to the
intended route of administration and standard pharmaceutical practice.
See for example, Remington's Pharmaceutical Sciences, 16th edition, Osol,
A. Ed., Mack Publishing Co., 1980.

[0147] Administration of a compound or composition of the invention by a
first administration route accompanied by separate, simultaneous or
sequential administration of one or more additional agents, including one
or more other antifungal agents, by the same or a second administration
route is contemplated; for example, oral or topical administration of a
compound or composition of the invention accompanied by oral or topical
administration of the at least one additional agent.

[0148] Also, the compositions useful herein may contain one or more
additional agents as required, including one or more additional
antifungal agents, or emulsifying, antioxidant, flavouring or colouring
agents, or have an enteric coating. Suitable enteric coatings are known.
Enteric coatings surrounding the active ingredients and prevent the
release of the active ingredients in the stomach bUt allow release after
the dosage form has left the stomach. Compositions useful herein may be
adapted for immediate, delayed, modified, sustained, pulsed or controlled
release of a compound of the invention.

[0149] Capsules can contain any standard pharmaceutically acceptable
materials such as gelatin or cellulose. Tablets can be formulated in
accordance with conventional procedures by compressing mixtures of the
active ingredients with a solid carrier and a lubricant. Examples of
solid carriers include starch and sugar bentonite. Active ingredients can
also be administered in a form of a hard shell tablet or a capsule
containing a binder, e.g., lactose or mannitol, a conventional filler,
and a tabletting agent. Parenteral dosage forms include aqueous
solutions, isotonic saline or glucose solutions comprising the active
agent, or other well-known pharmaceutically acceptable carriers.
Solubilising agents well-known to those familiar with the art can be used
as pharmaceutical excipients. Injectable dosage forms may be formulated
as liquid solutions or suspensions. Solid forms suitable for solution in,
or suspension in, liquid prior to injection may also be prepared.

[0151] Topical formulations may be prepared as lotions, creams, ointments,
pastes or salves using known carriers for such applications. Such
formulations may be administered directly, for example, applied directly
on to a site of infection, a wound, sprayed onto a surgical site, etc, or
may be applied indirectly, such as by impregnation into a bandage or
dressing or sprayed onto surgical equipment, dressings and the like.

[0153] The efficacy of a composition useful herein can be evaluated both
in vitro and in vivo. See, e.g., the examples below. Briefly, in one
embodiment the composition can be tested for its ability, to for example,
inhibit fungal growth in vitro. For in vivo studies, the composition can
be administered to an animal (e.g., a mouse) and its effects on fungal
infection, or one or more symptoms of the fungal disease or diSorder are
then assessed. Based on the results, an appropriate dosage range,
frequency, and administration route can be determined.

[0154] It should be understood that the additional agents listed above may
also be employed in a method of the invention where they are administered
separately, simultaneously or sequentially with a compound, isolate or
composition useful herein.

[0155] As will be appreciated, the dose of the composition administered,
the period of administration, and the general administration regime may
differ between subjects depending on such variables as the severity of
symptoms of a subject, the type of disorder to be treated, the mode of
administration chosen, and the age, sex and/or general health of a
subject. However, by way of general example, from about 1 mg to about
5000 mg per kg body weight, about 1 mg to about 4000 mg per kg body
weight, about 1 mg to about 3000 mg per kg body weight, about 1 mg to
about 2000 mg per kg body weight, about 1 mg to about 1000 mg per kg body
weight, or about 1 mg to about 500 mg per kg body weight of a compound
useful herein is administered, per administration or per day, preferably
about 50 to about 1000 mg per kg, preferably per day. Administration may
include a single dose, such as a single daily dose, or administration of
a number of discrete divided doses as may be appropriate. A person of
ordinary skill in the art will be able to determine without undue
experimentation, having regard to that skill and this disclosure, an
effective dosage regime (including dose and timing of administration) for
a given condition.

[0156] When used in combination with an additional agent, the
administration of a compound useful herein and the other agent may be
separate, simultaneous or sequential. Simultaneous administration
includes the administration of a single dosage form that comprises all
components or the administration of separate dosage forms at
substantially the same time. Separate or sequential administration
includes administration according to different schedules, preferably so
that there is an overlap in the periods during which the composition
useful herein and other therapeutic agent are provided.

[0157] Additionally, it is contemplated that a composition in accordance
with the invention may be formulated with additional active ingredients
which may be of benefit to a subject in particular instances. For
example, therapeutic agents that target the same or different facets of
the disease process may be used.

[0158] The compounds or compositions of the invention may be incorporated
into or onto medical devices and medical supplies. The medical devices or
supplies may be coated or impregnated with compositions of the invention
by known methods.

[0159] Various aspects of the invention will now be illustrated in
non-limiting ways by reference to the following examples.

EXAMPLES

1. Materials and Methods

(a) Fungal Strains

[0160] An Epicoccum purpurascens strain SVB-F1 was isolated in a
laboratory environment as a contaminant of other cultures. It was
identified based on its morphological characteristics, on its strong
anti-fungi activity against different filamentous fungi, and on the
sequence of its 18S rDNA (ITS region) (data not shown). The compound of
Formula (IV) was identified and isolated from the exudate of this strain.
Other Epicoccum purpurascens strains obtained subsequently and tested
were ICMP1732, ICMP2048, ICMP2931, ICMP10458, ICMP10459, ICMP10460,
ICMP11503, ICMP13352, ICMP15700, ICMP15816 and ICMP16305, all available
from Landcare Research. New Zealand. Active spore suspension and mycelia
on agar slants were kept preserved in glycerol saline at -80° C.

[0161] The phytopathogenic fungi Apiognomonia supraseptata SVB-F2 was
isolated from plant material and identified based on the sequence of its
18S rDNA (ITS region).

[0163] Strain SVB-F1 grows rapidly and produces woolly to cottony or felty
colonies on potato dextrose agar at 25° C. From the front, the
colonies are yellow to orange, orange to red or pink initially and become
greenish brown to black by aging. From the reverse, the same colour is
observed but is usually more intense than in the front view. Epicoccum
may produce a diffusible pigment which turns the colour of the inoculated
medium to yellow, orange, red or brown. Black dots (100-2000 μm in
diameter) may be observed macroscopically on the colony surface. These
are the tufts of hyphae which have conidiophores on their surface. These
tufts of hyphae are cushion-shaped and nonconvoluted and are called
sporodochia.

Metabolism

[0164] Strain SVB-F1 grows on a wide range of substrates and is able to
grow on minimal mineral medium with any carbohydrate as carbon source and
ammonia or nitrate as nitrogen source. Optimal growth temperature is
25° C. and the organism can tolerate a wide range of pH (3-10).

[0167] Production of antifungal pigment by E. purpurascens strain SVB-F1
was tested on three different complex culture media (CYA, YPD and PDA) as
well as on minimal mineral medium (MM) at pH 7 supplemented or not with
individual amino acids (5 g/L). All cultures were carried out using Petri
dishes incubated at 25° C. for 10 days.

(f) Extraction and Purification of the Biologically Active Compound

[0168] Ten day old E. purpurascens SVB-F1 cultures on agar plates were
diced and transferred to 250 mL Schott bottle containing 50 mL of
methanol. The flasks were shaken for 30 min at room temperature in the
dark. The methanol extracts were separated from the fungal biomass and
agar by filtration using cotton-wool followed by filtration using
nitrocellulose membrane (0.22 μm).

[0169] Pigmented compounds were pre-purified from the methanol extract by
solid-phase extraction using SPE DSC-Si silica 5 g cartridges
(Discovery®, Supelco, Bellefonte, USA). The cartridges were primed
with 3-volumes (20 mL) of deionised water, followed by 3- volumes (20 mL)
methanol, 3-volumes (20 mL) of acetonitrile and 1-volume (20 mL) of
acetonitrile/methanol mixture (1:1): Sample dissolved in
acetonitrile/methanol mixture (1:1) was eluted with the same solvent
mixture and the pigmented fraction was collected and dried under air-flow
in the dark.

[0170] The air-dried pigmented residues were washed with hexane to remove
non-polar compounds. Residual hexane was then removed by evaporation and
the precipitate resuspended in deionised water at pH 10. Pigmented
compounds were then further purified from the pre-purified extracts by
HPLC (Beckmann, USA) using UV detection at 360 nm. Pigments were
separated on a Gemini-NX C18 column (Phenomenex) at room temperature. Two
solvents, (A) deionised water at pH 10 (NH4OH), and (B)
methanol/isopropanol (4:1), were used in combination as the mobile phase
at a flow of 1 mL/min. A gradient from 0-12 min comprised 25% to 100%
(B), holding for 1 min and returning back to 25% over the next 7 min.
Pigmented fractions eluting from the column were collected and their
retention time recorded.

(g) Stability Tests

[0171] Stability was determined by re-suspending between 0.3 to 0.5 mg/mL
of purified pigmented compound of Formula (IV) in different testing
solutions according to Table 1, and measuring the absorbance at 428 nm
every 24 hours for fifteen days. The test samples were kept in glass
vials under constant incandescent light (except for the temperature
stability test).

[0172] The antagonistic properties of E. purpurascens SVB-F1 against other
filamentous fungi were assessed by co-culture on YPD plates incubated at
25° C. for 7 days in the dark. The fungi tested were Apiognoinonia
supraseptata SVB-F2, Botrytis cinera ICMP16621 (Landcare Research, New
Zealand) and Sclerotinia sclerotiorum ICMP13844 (Landcare Research, New
Zealand).

(i) Biological Activity Test--Inhibition of Mycelial Growth

[0173] The activity of the compound of Formula (IV) purified by HPLC was
tested using agar diffusion assays against mycelial growth of A.
supraseptata SVB-F2, B. cinera ICMP16621, and S. sclerotiorum ICMP13844.
The fungal strains were grown on YPD agar. Test compounds were absorbed
onto individual paper disks (6 mm diameter) or directly into the agar
through a 1-mm hole at 20 μL/disk of pigment solution in phosphate
buffer (pH 7.4). Phosphate buffer was used as a negative control. The
assay plates were incubated at 25° C. for 5 days and examined for
the presence of a zone of inhibition.

(j) Biological Activity Test--Inhibition of Spore Germination

[0174] The activity of the compound of Formula (IV) against the spore
germination of different phytopathogens was carried out in 12-well
microtitre plates containing 200 μL of fungal spore suspension
(˜106 spores.mL-1) and 1.8 mL of HPLC purified pigment resuspended
in sterile YPD broth.

[0175] Nine phytopathogenic fungi were tested: B. cinera ICMP16621, S.
sclerotiorum ICMP13844, Alternaria alternate ICMP1099-96, Phomopsis
viticola ICMP5537, Mycosphaerella graminicola ICMP12504-95, Rhizoctonia
solani ICMP 11620, Magnaporthe grisea ICMP14481Sclerotium cepivorwn
ICMP10916-91, and Venturia inaequalis ICMP4095-96. Three'different
concentrations of antifungal compound were tested: 2.7, 1.3 and 0.7
mg.mL-1 respectively. The cultures were incubated at 25° C. Each
treatment was prepared in triplicate. The germinated spores were observed
and recorded at 12, 24, 48, 72, 96, 120, and 240 hours. The percentage of
spores germinated was determined by microscopic examination of 3
microscopic fields (haemocytometer) per sample. Spores were considered
germinated when the germ tube length was equal or longer than the
diameter of the spore. YPD broth without antifungal compound was used as
positive control.

(k) Chemical Characterization

[0176] UV-Vis spectra were measured with a Hitachi High-Technologies
Corporation spectrophotometer model U1800. IR spectra were recorded on a
Thermo Electron Nicolet 8700 FT-IR spectrometer. A drop was placed on top
of the diamond crystal and left to dry for 1 hour to reduce the water
bands which dominated the spectrum, then 100 scans were collected at a
resolution of 4 cm-1 and averaged. The angle of incidence of the IR
beam was 45° and the spectrum was ATR-corrected and
baseline-corrected. Direct infusion electrospray FT-ICR MS analysis was
carried out on a Thermo LTQ-FT mass spectrometer in aqueous methanol in
positive ion mode. NMR spectra were recorded on a Bruker Avance 600
spectrometer equipped with a triple-resonance cryoprobe (Bruker,
Karlsruhe, Germany) at 600 MHz for 1H and 150 MHz for 13C in DMSO-d6. TMS
was used as an internal standard.

(l) Comparison of Different Strains of E. purpurascens

[0177] The Epicoccum purpurascens strains ICMP1732, ICMP2048, ICMP2931,
ICMP10458, ICMP10459, ICMP10460, ICMP11503, ICMP13352, ICMP15700,
ICMP15816 and ICMP16305 were all tested to determine whether they
produced the compound of Formula (IV) produced by Epicoccum purpurascens
strain SVB-F1. Each strain was cultured and extracellular products
extracted as described above. The culture extracts were pre-purified by
solid-phase extraction as described above and the purified extract were
analysed by HPLC and direct infusion electrospray FT-ICR MS as above.

(b) Properties and Production Oldie Compound of Formula (II), Named
"Epicoccaene"

[0180] E. purpurascens SVB-F1 when cultivated on protein-rich media
actively secretes a pigmented exudate. This pigmented exudate was
fractionated as described above and when tested according to the methods
described above showed a pronounced antifungal activity against
filamentous fungi. No antimicrobial activity was observed against yeasts,
Gram positive or Gram negative bacteria (data not shown).

[0181] E. purpurascens SVB-F1 can grow well in minimal mineral medium with
glucose and ammonium as respective sole carbon and nitrogen sources but
only produced the pigmented exudate when grown on media containing
proteinaceous substrates such as peptone, yeast extract, and potato
extract (data not shown). Growing E. purpurascens SVB-F1 on minimal
mineral agar medium supplemented with individual amino acids led to the
production of yellow pigment when histidine was supplemented to the
medium (data not shown).

[0182] The compound of Formula (IV) isolated from the pigmented exudate of
E. purpurascens SVB-F1, hereinafter "Compound 1" is readily soluble in
water and polar organic solvents such as methanol, and ethanol. It was
found to be unstable under acidic conditions changing from a bright
yellow colour to a pale orange. But when an acidic solution of Compound 1
was adjusted to pH 12, it recovered its bright yellow colour suggesting a
reversible pH-dependent structural rearrangement. Table 1 above
summarises the stability of Compound 1 to different conditions. It
exhibited pronounced degradation when dissolved in solution of organic
acids (10% w/w) as well as moderate light sensitivity when dissolved in
water at neutral pH. However, when dissolved in water at pH 8.0 or
higher, in methanol or in sugar solutions (10% w/w), its resistance to
light increased (Table 1). Compound 1 also appears to be resistant to
temperature and microwave radiation (Table 1), and its antioxidant power
determined by FRAP (ferric reducing antioxidant power) and
phosphomolybdenum assays as well as its radical scavenging ability are
comparable to those of ascorbic acid (data not shown).

(c) Chemical Characterisation

[0183] Compound 1 has the molecular mass of 612 and the molecular formula
of C34H45O10 from ES/FT-ICR/MS (found 613.29973 for MH+,
C34H45O10) measurements, indicating that Compound 1 is an
isomer of orevactaene, a binding inhibitor of HIV-1 Rev protein also
isolated from an E. purpurascens culture (Shu et al, 1997). However the
maximal UV absorption of the purified compound was obtained at 428 nm
suggesting a slightly different conjugation system compared to
orevactaene which presents UV. max at 432. The IR spectrum of Compound 1
showed the strong absorption characteristics of hydroxyls (3375.5 and
3220.0 cm-1), hydroxyls linked to carbonyls (2903.6-3023.0
cm-1), conjugated carbonyl (1676.8 cm-1), and additional
absorptions suggestive of the presence of an ester/lactone (1093.9-1204.9
cm-1) and alkenes (1005.1 cm-1). NMR and 13C NMR spectra
of Compound 1 were almost identical with those of orevactaene (data not
shown). However, larger than average chemical shift differences were
observed for C1, C2, C8 and C7, which indicate structural differences
between Compound 1 and orevactaene. Based on HMBC, HSQS and COSY
connectivity data, the structure of Compound 1 was determined to be
Formula (IV) described. above:

##STR00010##

[0184] Compound 1 possesses two major structural differences compared to
orevactaene. Firstly, Compound I has a carboxyl group at C 1, which
explain the chemical shift differences of C1, C2, C8 & C7. Secondly,
despite not dramatically influencing the resonance of C3 and C33, it is
postulated that the oxygen bond to these carbons is linked forming a
seven-membered peroxide ring because this is the only way to match the
molecular mass of 612 and the corresponding fragmentation pathways
determined by high-resolution mass spectrometry shown in the
representation of the compound of Formula (IV) below.

##STR00011##

[0185] Also, eleven double bonds are present in the proposed structure for
Compound 1, which is close to 12.5 predicted by high resolution MS. The
stereochemistry of the chiral centres at C4, C5, C32 and C33 could not be
determined unambiguously with current data. Further NMR experiments are
required.

[0187] Compound 1 was also ableto completely inhibit spore germination of
B. cinera during incubation in rich culture medium for 10 days (data not
shown). In comparison, B. cinera spores were fully germinated within 12
hours of incubation in the positive control samples, which demonstrates
the strong inhibition effect of Compound 1 (data not shown). All three
different concentrations of Compound 1 tested were able to completely
inhibit spore germination not only of B. cinera, but also of S.
sclerotiorum ICMP13844, A. alternata ICMP1099-96, P. viticola ICMP5537,
R. solani ICMP11620, M. grisea ICMP14481, S. cepivorum ICMP10916-91, and
V. inaequalis ICMP4095-96; which indicate that the minimal inhibition
dose (MID) of compound 1 against these fungi is below 0.7 mg/mL. Only M.
graminicola ICMP12504-95 was resistant to all concentrations of Compound
1 tested.

(f) Chemical Comparison of Different Strains of E. purpurascens

[0188] Eleven additional E. purpurascens strains were subsequently
obtained to determine whether they produced Compound 1. Within the
elevenE. puipurascens strains studied, two were isolated outside New
Zealand (ICMP10458 ["overseas"] and ICMP13352 [United Kingdom]) and nine
were isolated from different parts of New Zealand. Only two E.
purpurascens strains, ICMP13352 and ICMP1732, failed to produce Compound
1. No pigment production was observed during growth of these strains
either.

INDUSTRIAL APPLICABILITY

[0189] The compounds, isolates, compositions and methods of the invention
are useful to treat or prevent fungal infections, particularly
filamentous fungal inventions in animals and plants, alone or delivered
separately, simultaneously or sequentially with another pharmaceutically
or agriculturally acceptable agent.